QUANTITATIVE ANALYSIS OF REDUCING SUGARS IN SUGAR PREPARATIONS

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JCAM No.114-R1

Japan Customs Analysis Methods No. 114

Quantitative Analysis of Reducing Sugars in Sugar Preparations consisting of Sugar and Dextrin (Issued in June 1999) (Updated in May 2001)

water.

1. Scope This

analysis

method

is

applied

to

sugar

3.2. Preparation of standard dextrose solution

preparations which consist of sugar and dextrin and

for standard-addition

which require the determination of their “reducing

Accurately weigh about 4 g of dextrose, transfer to a

sugar

contents,

expressed

as

dextrose

on

dry

substances,” as referred to in Note 2 to Chapter 35 in

Customs Tariff Law (Appendix Table–Customs Tariff

100 mL volumetric flask, and dilute to volume with water.

Schedule).

3.3. Preparation

of

standard

2. Outline of Test Method

(1) Standard invert sugar solution

invert

sugar

solution and other reagents Accurately weigh 4.75 g of sucrose, transfer

This analytical method is applied for products

with 90 mL of water to a 500 mL volumetric flask,

containing sucrose and dextrin (starch degradation

and add 5 mL of hydrochloric acid (specific gravity,

products) to determine reducing sugars, e.g. dextrose

1.18). After leaving to stand at 20–30°C for three

and maltose, contained in the dextrin. The procedure is

days, dilute the solution to volume with water and

summarized below.

store in a cool dark place.

(1) Determination of moisture content

Transfer a 50 mL portion of the solution above

(2) Determination of direct reducing sugars by the

to a 200 mL volumetric flask, neutralize with 1

Lane-Eynon method

mol/L sodium hydroxide aqueous solution using

(3) Determination of sucrose by the Lane-Eynon

phenolphthalein as an indicator, and dilute to

method

volume with water.

(4) Determination of the content of dextrin

Use the solution as standard invert sugar

(5) Calculation of DE value

solution for the standardization of Fehling’s Solution.

3. Reagents All chemicals must be JIS special reagent grade or

(2) 1% Methylene Blue solution Dissolve 1 g of methylene blue in water to

equivalent, unless otherwise specified.

make 100 mL. (3) Fehling’s Solution

3.1. Preparation of standard dextrose solution for making a calibration curve Accurately weigh about 1 g of dextrose, transfer to a 1,000 mL volumetric flask, and dilute to volume with

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Solution A:

Dissolve 34.639 g of copper sulfate (CuSO4•5H2O) in water to make

JCAM No.114-R1 exactly 500 mL, leave it for two days,

3.5.3. Invertase solution

and then filter.

Dissolve invertase in the 0.1M acetic acid buffer

Solution B: Dissolve 173 g of potassium sodium tartrate (KNaC4H4O6•4H2O) and 50g of sodium hydroxide in water to make exactly 500 mL, leave it for two days,

(pH 4.6) so that the concentration of invertase becomes 400 units/mL.

3.5.4. Enzyme solution for determination of dextrin

and then filter.

Dissolve

glucoamylase

(1,4-α-D-Glucan

glucohydrolase EC 3.2.1.3) and α-amylase (α-1,4-Glucan

3.4. Standardization of Fehling’s Solution Put 5.0 mL of Fehring’s Solution A and 5 mL of

4-glucanohydrolase EC 3.2.1.1) in the 0.2M acetic acid

Fehring’s Solution B into a 200 mL Erlenmeyer flask

buffer (pH 4.8) so that their concentrations become 20

containing a few glass beads and add from a 50 mL

units/mL(3) and 80 units/mL,(4) respectively. (5)

burette 19.5 mL of the standard invert sugar solution. After boiling it on an electric stove (heater) for two

Note 3) One unit represents the amount of enzymes able to produce 10 mg of glucose from soluble

minutes, add four drops of the methylene blue solution.

starch (as substrate) every 30 minutes at 40°C

Complete titration within a total boiling time of three minutes by dropwise addition of the standard invert sugar solution—without preventing boiling—until the

and pH 4.5. Note 4) One unit represents the amount of enzymes able to produce 0.18 mg of reducing sugar,

blue color disappears. Repeat titration twice and

expressed as glucose, from soluble starch (as

calculate the mean of three parallel titrations. (1) Obtain the factor of the Fehring’s Solution from the following formula:

substrate) per minute at 40°C and pH 6.0. Note 5) When using enzymes whose potencies or units have been determined based on different

Factor (2) = 20.36 / A

definitions, confirm in advance that their recovery rates in digesting 50 mg of corn starch

Where– A: Volume (mL) of the standard invert

are 100%.

sugar solution required Note 1) Use the mean value of the three parallel

3.5.5. Glucose determination kit

titrations as “A”; duplicate titrations must agree to within 0.1 mL in the volume of the sugar solution required Note 2) Calculate factor by rounding off fractions to the

Use a commercially available enzyme-based assay kit for the determination of glucose (dextrose).

3.6. Deproteinizing agent Solution A: Dissolve

third decimal place; the factor must be within a

in 100 mL of water and adjust the pH to 4.6 using a 5% sodium hydroxide aqueous solution. Transfer the solution to a 1,000 mL volumetric flask and dilute to

of

zinc

sulfate

Solution B: Dissolve 1.8 g of barium hydroxide

3.5. Preparation of buffers and enzyme solutions

Weigh 6 g of glacial acetic acid in a beaker, dissolve

g

(ZnSO4•7H2O) in 100 mL of water.

range of 1±0.02.

3.5.1. 0.1M acetic acid buffer (pH 4.6)

2

[Ba(OH)2•8H2O] in 100 mL of water.

4. Preparation of samples Prepare and collect analysis samples in appropriate manners,

e.g.

sample

reduction

methods,

etc.,

depending on their conditions presented. For powder or

volume with water.

crystal mixtures, grind them with a grinder or a mixer.

3.5.2. 0.2M acetic acid buffer (pH 4.8)

in mortars. In any case, collect relatively large amounts

For pasty or wet materials, homogenize them by mixing

By adding 120 mL of a 0.2M sodium acetate to 80 mL of 0.2M acetic acid, adjust the pH of the mixed solution to 4.8.

of samples randomly, and grind or mix them to uniformity.

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JCAM No.114-R1 compositions, change the amount of sucrose

5. Procedure

added so that the sucrose concentrations in the

5.1. Determination of moisture content

test solution and the blank solution become

Accurately weigh about 2 g of the sample homogenized in 4. in a weighing bottle which has been previously dried to a constant weight. Dry it in a vacuum oven at a temperature of 70–75°C for four hours, cool to room temperature in a desiccator and weigh. Repeat vacuum drying until the loss in weight does not exceed 2 mg per hour in the drying period. Calculate the moisture content in the sample from the following formula. Round off fractions to the first decimal place.

almost the same.

5.3.3. Titration Put 5.0 mL of Fehring’s Solution A and 5 mL of Fehring’s Solution B into a 200 mL Erlenmeyer flask. Add from a 50 mL burette 15 mL of the test solution prepared in 5.3.1., and titrate as described in 3.4 (preliminary titration). Then, put 5.0 mL of Fehring’s Solution A and 5 mL of Fehring’s Solution B into another 200 mL Erlenmeyer flask. Add from a 50 mL burette the test solution within 1 mL of the anticipated end point from the result of the

%, moisture content = {(W0 − W1 ) / W0 }× 100

preliminary titration above, and titrate in the same manner as in the preliminary titration.

Where–

Multiply by the factor of the Fehling’s Solution the

W0: Amount (g) of sample collected

volume (mL) of the test solution required in order to

W1: Weight (g) of sample after drying

obtain

the

corrected titre,

concentration of

5.2. Preparation of sample solution Accurately weigh 15 g of the sample homogenized in 4., and dissolve in water. Transfer the solution to a 500 mL volumetric flask and dilute to volume with

the

X

(mL).

Obtain

the

direct reducing sugars,

Ds

(mg/100mL) from the titre, X (mL), by reference to the appended Lane-Eynon’s Table (dextrose). Similarly, perform titration with the blank solution and obtain the

water.

concentration of the direct reducing sugars, Ds’

5.3. Determination of Direct Reducing Sugar in

making reference to the appended Lane-Eynon Table

Sample

(mg/100mL), from the corrected titre, X’ (mL), by (dextrose). Using Ds and Ds’, calculate the content (%) of direct

5.3.1. Preparation of test solution Put a 100 mL portion of the sample solution

reducing sugar from the following formula:

prepared in 5.2. in a 200 mL volumetric flask, add 10 mL

of

the

standard

dextrose

solution

for

standard-addition prepared in 3.2., and dilute to volume

%, direct reducing sugar =

with water.

S

100

100

Where– Ds: Concentration (mg/100mL) of direct reducing

5.3.2. Preparation of blank solution Put a 10 mL portion of the standard dextrose solution for the standard-addition prepared in 3.2 in a 200 mL volumetric flask, add about 2.5

g(6)

of sucrose

and dissolve by adding a small amount of water. Dilute the solution to volume with water. added specifically for samples consisting of

83 % of sucrose and 17 % of dextrin. Thus, analyzing

samples

sugar in test solution, obtained by reference to

the

with

different

3 of Page 6

appended

Lane-Eynon

Table

(dextrose). Ds’: Concentration (mg/100mL) of direct reducing sugar

Note 6) This is a reference on the amount of sucrose

when

(Ds − Ds' ) × 500 × 200 × 100

in

blank

solution,

obtained

by

reference to the appended Lane-Eynon’s Table (dextrose). S: Weight (mg) of sample collected in 5.2.

JCAM No.114-R1 (Ref.) Dilution rate =

DRs: Concentration

500 200 × 100 100

(mg/100mL)

of

direct

reducing sugar in test solution, obtained by reference to the appended Lane-Eynon Table [Invert sugar (without sucrose)].

5.4. Determination of sucrose in sample

DRs’: Concentration

5.4.1. Inversion reaction

(mg/100mL)

of

direct

reducing sugar in blank solution, obtained

Put a 20 mL portion of the sample solution

by reference to the appended Lane-Eynon’s

prepared in 5.2. into a 200 mL volumetric flask and add

Table [Invert sugar (without sucrose)].

1 mL of the invertase solution. Hydrolyze the sample

S:

solution by placing the flask in a water bath at a

Weight (mg) of sample collected in 5.2.

constant temperature of 37°C for 30 minutes, add 5 mL each of the deproteinizing solutions A and B, and mix

%, Sucrose content = {invert sugar content (%) –

thoroughly. Dilute the deproteinized solution to volume

A (%)} × 0.95

with water and filter. Use the filtrate as test solution for the determination of sucrose.

5.4.2. Titration

and

Note 7) The reason why two types of direct reducing

calculation

of

sugar contents in dextrin are calculated is that

sucrose

the content of direct reducing sugar, expressed

content

as invert sugar, is needed for the determination

Titrate with the test solution prepared in 5.4.1. in

of sucrose content, whereas that expressed as

accordance with the procedure in 5.3.3. Obtain the

dextrose is needed for the determination of DE

concentration of the invert sugar, T (mg/100mL), by

value.

reference to the appended Lane-Eynon’s Table [invert sugar (without sucrose)] and calculate the invert sugar

5.5. Determination of dextrin content

content (%) from the following formula.

5.5.1. Enzymatic digestion

%, invert sugar =

Put a 2 mL portion of the sample solution prepared

T 500 200 × × × 100 S 20 100

in 5.2. in a 100 mL volumetric flask and add 5 mL of the enzyme solution for determination of dextrin. Place the

Where–

flask in a water bath at a constant temperature of 37°C

T: Concentration (mg/100mL) of invert sugar in test solution, obtained by reference to the appended Lane-Eynon’s Table [invert sugar (without sucrose)].

for two hours for enzymatic digestion, add 5 mL each of the deproteinizing solutions A and B, and mix thoroughly. Dilute the solution to volume with water and filter. Use the filtrate as test solution for the determination of dextrin.

S: Weight (mg) of sample collected in 5.2. Next, from the titres (X and X’) obtained in 5.3.3,

5.5.2. Preparation

obtain the contents of the direct reducing sugar in the sample and blank solutions, DRs (mg/100mL) and DRs’ (mg/100mL), by reference to the appended Lane-Eynon Table [Invert sugar (without sucrose)]. Calculate from the following formula the content of the direct reducing sugar, expressed as invert sugar, in dextrin contained in the

A (%) = Where–

100

100

curve

for

dextrose solutions prepared in 3.1. in 100 mL volumetric flasks, respectively, and dilute the solutions to volume with water. Use them as standard solutions for constructing a calibration curve. Using

S

calibration

Put 5, 10, 15, 20 and 25 mL of the standard

sample, A (%).(7)

(DRs − DRs') × 500 × 200 × 100

of

dextrose

a

commercially

available

dextrose

determination kit, e.g. glucoxidase-peroxidase-based assay kits, described in 3.5.5., prepare a calibration curve

by

plotting

absorbance

against

dextrose

concentration (mg/ml) for each of the standard solutions

4 of Page 6

JCAM No.114-R1 prepared

above.

Construct

the

calibration

curve

simultaneously during the procedure in 5.5.3.

5.5.3. Determination of dextrose calculation of dextrin content

6. References (1) 中村道徳, 貝沼圭二 「澱粉・関連糖質実験法」学術出 版センター (1986)

and

(2) 三国二郎 監修 「澱粉化学ハンドブック」 朝倉書店

Utilizing the same assay kit described in 3.5.5., quantify dextrose in the test solution prepared in 5.5.1. and determine the concentration (mg/ml) of dextrose in the test solution using the calibration curve constructed in 5.5.2. Calculate the dextrin content (%) in the sample from the following formula.(8, 9)

%, dextrin =

Dextrose (%) × 0.9 × Dilution rate × 100 S

Where– S: Note 8)

Amount (mg) of sample collected in 5.2. In the calculation, the dextrin content is deemed to be equal to “dextrose (%) × 0.9.”

Note 9)

Dextrin content may be calculated as a balance. In that case, calculate the dextrin content from the following formula: Dextrin content (%) = 100 – {sucrose content (%) +moisture content (%)} However, when the calculated value looks doubtful due to the differences from those in the

attachments

and

composition

table

provided, determine the exact value with assay.

5.6. Calculation of DE Calculate the content of reducing sugars, expressed as dextrose on the dry substance, in dextrin contained in the sample by applying the following formula with the values obtained in 5.3.3. and 5.5.3. Round off fractions to the first decimal place. DE =

Direct reducing sugar content (%) in dextrin × 100 Dextrin content (%) in sample

Where– DE: Content (%) of reducing sugar, expressed as dextrose on the dry substance, in the dextrin contained in the test sample.

5 of Page 6

(1977)

JCAM No.114-R1 Appendix Lane-Eynon Table (Invert sugar and Dextrose) Saccharides mL sugar solution required

Invert sugar (without sucrose) mg/100ml

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

336 316 298 282 267 254.5 242.9 231.8 222.2 213.3 204.8 197.4 190.4 183.7 177.6 171.7 166.3 161.2 156.6 152.2 147.9 143.9 140.2 136.6 133.3 130.1 127.1 124.2 121.4 118.7 116.1 113.7 111.4 109.2 107.1 105.1

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Dextrose (anhydrous) mg/100ml 327 307 289 274 260 247.4 235.8 225.5 216.1 207.4 199.3 191.8 184.9 178.5 172.5 167 161.8 156.9 152.4 148 143.9 140 136.4 132.9 129.6 126.5 123.6 120.8 118.1 115.5 113 110.6 108.4 106.2 104.1 102.2